Stubomycin Was Isolated from the Culture Broth and Mycelia of Streptomyces Strain No. KG-2245 by UMEZAWA Et Al. in 1981.1) Stubo

VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 301 STUDIES ON THE BIOLOGICAL ACTIVITY OF STUBOMYCIN KANKI KOMIYAMA,KEN-ICHi EDANAMI, AKIRA TANOH, HIROSHI YAMAMOTOand IWAO UMEZAWA The Kitasato Institute, Minato-ku, Tokyo 108, Japan (Received for publication November 30, 1982) Stubomycin showed direct cytotoxic activity on mammalian cells, yeast, and fungi, and rapid hemolytic activity on mouse erythrocytes. The rate and extent of the cytotoxic and hemolytic activities decreased at lower temperatures. Studies with radioactive precursors revealed that a marginal cytocidal concentration of the antibiotic inhibited synthesis of DNA, RNA, and protein of leukemic cells at almost the same rate. Stubomycin did not show any mutagenicity on mammalian cells and bacteria i.e. the induction of revertants on six bacterial strains, and chromosomal aberrations, sister chromatid exchanges, and the induction of cells resistant to 6-thioguanine on Chinese hamster cells (DON D-6). The antagonistic effect of various kinds of lipids including phospholipids, cholesterol, olive oil and squalene was studied. Significant antagonism of stubomycin against anti-Saccharomyces cerevisiae activity was observed with phospholipids except for egg lecithin and with cholesterol. The primary action of the antibiotic seems to be to change the cell surface and ultimately the lysis and death of the cells. Stubomycin was isolated from the culture broth and mycelia of Streptomyces strain No. KG-2245 by UMEZAWAet al. in 1981.1) Stubomycin has been shown to have marked in vivo antitumor activity on Ehrlich ascites carcinoma, P 388 leukemia, Meth A fibrosarcoma, etc. The antibiotic is also active against Gram-positive bacteria, some fungi, and HeLa cells in vitro.') Recently, OMURAet al. have eluci- dated the structure of stubomycin as a macrocyclic lactam involving (3-phenylalanine.3) This report describes preliminary studies on the cytocidal activity of stubomycin mainly using mammalian cells, yeast, and fungi. Materials and Methods Chemicals N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG, Nakarai Chemicals Ltd., Kyoto), 2-nitrofluorene .(Aldrich Chemical Co ., USA), and 9-aminoacridine (Tokyo Kasei Kogyo Co., Tokyo) were dissolved in dimethyl sulfoxide. Ethyl methanesulfone and 6-thioguanine (EMS and 6-TG, Sigma Chemical, USA), mitomycin C (Kyowa Hakko Kogyo Co., Tokyo), and benzylpenicillin (Toyo Jozo Co., Shizuoka) were dissolved in phosphate buffer or water. [3H]Thymidine ([3H]TdR, 16.2 Ci/mmole), [3H]uridine ([3H]UR, 38.3 Ci/mmole), and [3H]leucine (158.0 Ci/mmole) were obtained from the Radiochemical Center, Amersham, UK. Cardiolipin, pho- sphatidylserine, and egg lecithin (Kitasato Institute, Tokyo), olive oil and cholesterol (Wako Pure Chemi- cal Industries, Ltd., Osaka), and squalene (Kuraray Co., Osaka) were dissolved in ether or methanol. Cells and Media Salmonella typhimurium strain numbers TA98, 100, 1535, 1537, and 1538, and Escherichia coli WP 2uvrA were provided by Dr. T. KADA,National Institute of Genetics. DON D-6 cells, derived from Chinese hamster lung cells, were maintained in EAGLE'Sminimum essential medium (MEM) supplemented with fetal calf serum (10 %), sodium pyruvate (1 MM), L-serine (0.2 mm), penicillin (100 u/mI), and streptomycin (100,ag/ml) at 37°C in an atmosphere of 5 % CO, in air. Mouse leukemic EL-4 cells 302 THE JOURNAL OF ANTIBIOTICS MAR. 1983 were maintained in MEM supplemented with 10 % calf serum, and cells in the logarithmic growth phase were used for the experiment. Saccharomyces cerevisiae and Pyricularia oryzae have been maintained in our laboratory on potato agar and rice straw agar respectively. To determine the effect of stubomycin on these microorganisms, two media were used: (1) 0.3 % yeast extract, 0.3 % peptone, 3 % glucose, and 0.3 % Trypticase soy broth for S. cerevisiae; and (2) 0.5 % peptone, 0.3 % yeast extract, and 3 % glucose-potato extract solution (200g of potato was boiled in 1 liter of water for 30 minutes, and the solution was used as potato extract) for P. oryzae. Measurement of Cytotoxicity To determine the cytotoxicity of stubomycin on mammalian cells, EL-4 cells (2 x 106) in 2 ml of medium were placed in 30-mm Petri dishes and incubated for 48 hours. Then, 0.01 ml of different concentrations of stubomycin solution were added to each culture dish, and thereafter, the number of trypan blue excluded cells was counted periodically using a hemocytometer. In case of antibacterial activity, the antibiotic was added to suspensions of S. cerevisiae (optical density: 0.092) and P. oryzae (optical density: 0.043) in 4 ml of medium, and the mixtures were shaken at 27°C. The growth of S. cerevisiae was followed by determining the absorbancy of cultures at 660 nm. In case of P. oryzae, the culture was gently dispersed by a homogenizer (Biotron), and the growth of P. oryzae was followed by determining the absorbancy of cultures at 660 nm. Values are the mean of three samples. Measurement of Viability EL-4 cells (7 x 105) in 7 ml of MEM were exposed to 0.05 ml of different concentrations of stubomycin solution for 5 to 60 minutes. Then at the end of each incubation time, cells were rinsed three times with fresh MEM, and were suspended in 6 ml of culture media. Two ml of each cell suspension were plated on 30-mm Petri dishes. Viable cells were counted after incubation at 37°C for 72 hours. For viable count determination on S. cerevisiae, the microorganism suspended in medium was exposed to different concentrations of stubomycin for 10, 30, or 60 minutes. After incubation, the cells were washed three times with 5 ml of ice-cold medium by centrifugation, diluted with the medium, and plated on potato dextrose agar. After 48 hours of incubation at 37°C, the colonies were counted. Effect of Temperature on Anti-S. cerevisiae Activity of Stubomycin S. cerevisiae (optical density: 0.24) was exposed to 1.25 ,ug/ml of stubomycin for 30 minutes at different temperatures. At the end of incubation, the cells were centrifuged at 0°C (600 x g, 2 minutes). The cells were then resuspended in the growth medium. After incubation for 6.5 hours at 27°C, the absorbancy of the cultures was determined at 660 nm. Measurement of Macromolecular Synthesis EL-4 cells (1 X 106 in 2 ml of MEM) were mixed with stubomycin and various precursors, and incubated at 37°C for 60 minutes. The cells were then collected on a Millipore filter and rinsed 3 times with ice-cold 5 % trichloroacetic acid. The radioactivity of acid-precipitable material on the filter was determined by a Packard Tri-Carb liquid scintillation spectrometer. Mutagenicity of Stubomycin Effect of Stubomycin on Induction of Revertants in Six Bacterial Strains: The method for detec- tion of mutagenicity as described by AMESet al.') and YAHAGiet al.5>was used. Briefly, stubomycin was dissolved in DMSO and diluted with sterilized distilled water. The antibiotic solution (0.1 ml) was mixed with or without 0.5 ml of S-9 solution, and these mixtures were combined with 0.1 ml of bacterial suspension (1 x 109), then incubated at 37°C for 20 minutes. After incubation, 2 ml of soft agar were added to the mixture and the mixture was plated on 90-mm Petri dishes. After 2 days of incubation at 37°C, the number of revertant colonies were counted. As positive mutagen controls, 2-aminoanthracene for the metabolic study and ENNG, 2-nitrofluorene, and 9-aminoanthracene for the non-metabolic study were used according to the bacteria applied as test organism. DNA Damaging Effect of Stubomycin Determined by the rec-Assay: The rec-assay with Bacillus subtilis was performed by the method described by KADAet al.°'7> Briefly, strains of M 45 or H 17 were VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 303 cultured overnight in brain heart infusion broth. Each culture was streaked radially on the surface of broth agar, and a paper disk (diameter: 8 mm) containing the drug test solution was placed over the starting point of the streaks. The plates were kept at 4°C for 30 minutes, then incubated overnight at 37°C. The lengths of the inhibitory zones were measured. Effect of Stubomycin on Chromosomal Aberrations and Sister-chromatid Exchanges in DON Cells : DON cells (1 x 101) in 5 ml of medium were plated on 60-mm Petri dishes and incubated at 37°C for 15 hours. After plating, the cells were exposed to stubomycin or EMS for 31 - 50 hours in medium with or without 5'-bromodeoxyuridine (10-1 M). Three hours prior to harvest, Colcemide was added to give a final concentration in the culture of 0.05 µg/ml. At the time of harvest, the trypsinized cells were gently centrifuged, resuspended in hypotonic saline (0.075 M KCI) and allowed to stand for 10 minutes. After fixation with a mixture of methanol - acetic acid (3: 1), the cell suspensions were dropped onto slides, allowed to air dry and stained with Giemsa. Effect of Stubomycin on Induction of Mutation Resistant to 6-TG in DON Cells: DON cells (2 x 101) in 4.5 ml of medium were plated in 25-cm' plastic flasks (Corning, USA). After plating, 0.5 ml of the antibiotic or EMS solution was added to the culture which was then reincubated for 15.5 hours. The resulting culture was washed with phosphate buffered saline, resuspended in fresh MEM, and further incubated for 8.5 hours (day 0). To express mutation, the cells were subcultivated every other day, and the cells were trypsinized to select the mutants on days 13 or 15. The resulting cells (5 x 104) in 10 ml of medium containing 6-TG (10-1 M) were plated on 100-mm Petri dishes.

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